ABSTRACT: Over 85% of patients with advanced cancers of breast and prostate suffer from incurable bone metastases. Osteoclast activation and osteoblast suppression lead to bone loss, pathologic fractures, hypercalcemia and bone pain, often accompanied by loss of skeletal muscle (cachexia). Current therapies target osteoclasts and have limited effects on tumor growth, while not restoring lost bone or muscle. Tumor-secreted parathyroid hormone-related protein, PTHrP, is a major causal agent of bone destruction by inducing expression of RANKL to increase osteoclast number and function. TGF?, released during bone resorption, stimulates breast cancer production of PTHrP [Yin et al, 1999], driving a vicious cycle of bone metastases [Chirgwin & Guise, 2000; Kakonen et al, 2002]. PTHrP stimulates growth of PTH1R receptor- expressing tumors and indirectly causes cancer cachexia via reprogramming of adipocytes [Kir & Spiegelman, 2016]. It causes hypercalcemia of malignancy, stimulates angiogenesis and cancer bone pain. We believe the deleterious actions of tumor PTHrP can be blocked by a unique PTH1R ligand, while having beneficial anabolic effects on bone - which cannot be achieved with PTHrP-neutralizing antibody. [D-Trp12,Tyr34]-bovine-PTH(7- 34), bPTH7-34DD, is a unique inverse agonist of G protein-coupled signaling by PTH1R, decreasing cAMP, while activating ?-arrestin signaling. It increases bone formation without causing hypercalcemia or osteolysis [Maudsley et al, 2015]. Our data showed anti-osteolytic and anti-tumor actions of bPTH7-34DD in co-cultures of breast cancer with mouse bone. It also countered adipocyte gene changes associated with cancer cachexia. We hypothesize that bPTH7-34DD will: 1) Inhibit the vicious cycle of osteolytic bone metastases by suppressing osteolysis & tumor growth; 2) Decrease cancer cachexia by blocking white-to-brown adipocyte conversion in bone; 3) Preserve bone health by stimulating new bone formation; 4) Effectively treat osteolytic tumors due breast cancer. 5) Be effective against other PTHrP-secreted tumor types. bPTH7-34DD will be tested in xenograft models of breast cancer bone metastasis, in prevention and treatment settings. The mechanism of bPTH7-34DD signaling will be tested in ex vivo co-cultures of cancer cells with mouse bones and in bone and fat cell lines and 1o cells and with bones and cells from wt & ?-arrestin k/o (?arr2-/-) mice. Specific Aim 1: a) Determine growth effects ex vivo of bPTH7-34DD on tumor cells; b) Determine the mechanism of action of on i) Osteoblasts (Obs), osteoclasts (Ocs), & osteocytes (Ots); ii) On adipocytes; c) Test if responses to bPTH7-34DD require ?-arrestin, using bones & cells from ?arr2-/- mice; d) Test if bPTH7-34DD blocks growth in bone and osteolysis ex vivo with prostate and lung cancer and multiple myeloma cells Specific Aim 2: Test bPTH7-34DD in vivo for significant effects on: a) Tumor burden; b) Osteolytic lesions; c) Bone loss/formation systemically and adjacent to tumor; d) Skeletal muscle. Significance: bPTH7-34DD could be rapidly brought into clinical trials for cancer bone metastasis. Similar PTH & PTHrP peptides are used to treat osteoporosis. Benefit may extend to other PTHrP-expressing cancers that metastasize to bone, including multiple myeloma, which also colonizes bone and causes osteolysis. Technical Approaches: The project uses co-cultures of cancer cells with mouse bones, called EVOCA (ex vivo organ co-culture assay). Tumor growth is assayed by secreted Gaussia luciferase (gLuc) from stably transduced cancer cells. Changes in bone and MM genes are quantified by mouse- and human-specific qPCR. Two mouse models of breast cancer bone metastases will be used for preclinical testing. Consultants: Dr. Louis Luttrell is a world expert in PTH signaling and deciphered the actions of bPTH7-34DD. Dr. Teresa Zimmers is an expert in cancer cachexia. Dr. Jesus Delgado-Calle is an expert in osteocyte biology and osteocyte:adipocyte interactions in myeloma. Dr. David Roodman is a world expert on myeloma bone disease.